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Novel anti-human Tie2 antibody

An antibody and antigen technology, applied in the direction of antibodies, antibody medical components, antibody mimics/scaffolds, etc., can solve problems such as increased retinal cell damage

Active Publication Date: 2017-05-17
ASTELLAS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Meanwhile, increased retinal cell damage has been reported in genetically modified mice with Ang-2-specific overexpression in the retina (Acta. Diabetol. 2010, Vol. 47, pp. 59-64)

Method used

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  • Novel anti-human Tie2 antibody
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  • Novel anti-human Tie2 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0246] (Example 1: Preparation of hybridoma producing anti-human Tie2 antibody)

[0247] Antibodies were prepared using "VelocImmune" (VelocImmune Antibody Technology: Regeneron Corp. (US Patent No. 6596541)) - Human Monoclonal Antibody Development Technology - Mouse. Recombinant human Tie2-Fc chimeric protein (R&D, 313-TI-100) was injected into VelocImmune mice together with adjuvant for immune response for immunization. According to the usual method, lymph nodes of immunized mice were extracted, and lymphocytes were collected and subjected to cell fusion with mouse-derived myeloma cells SP2 / 0 (ATCC: CRL-1581), thereby preparing hybridomas. The hybridomas were monoclonalized, and each clone was cultured in CD Hybridoma Medium (Invitrogen) as a serum-free medium. Antibodies were purified from the obtained culture supernatant using a protein G column (GE Healthcare). The antibody obtained using the VelocImmune technology is an antibody having a variable region of a human anti...

Embodiment 2

[0248] (Example 2: Cell ELISA Assay)

[0249] In order to measure the antigen-binding activity of the antibody, human Tie2-expressing CHO cells, monkey Tie2-expressing CHO cells, rat Tie2-expressing CHO cells, and mouse Tie2-expressing CHO cells were each evaluated by cell ELISA assay. Antibody binding to human Tie2, monkey Tie2, rat Tie2 and mouse Tie2.

Embodiment 3

[0250] (Example 3: Evaluation of Competitive Activity Using Modified Ang-1)

[0251]In order to assess the Ang-2 competitive activity of the antibody, the modified Ang-1 (Proc.Natl.Acad.Sci., 2004, Vol.101, pp.5547-5552, also known as COMP-Ang1) and Inhibition of binding of Tie2. COMP-Ang1 is a modified Ang-1 in which the site not involved in binding to Tie2 is modified, and the binding ability of COMP-Ang1 to Tie2 is maintained (Proc.Natl.Acad.Sci.2004, Vol.101 , pp.5547-5552), and Ang-1 and Ang-2 bind to the same site of Tie2 with the same affinity level (Science, 1997, Vol.277, pp.55-60) point of view, it resists The competitive effect of Ang-2 can be assessed by assessing the competitive effect against COMP-Ang1.

[0252] The expression vector of COMP-Ang1 was introduced into HEK293 cells. COMP-Ang1 was purified from the culture supernatant of HEK293 cells and biotin-labeled. The biotin-labeled COMP-Ang1 was mixed with the purified antibody obtained in Example 1, and t...

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PUM

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Abstract

[Problem] To provide an anti-human Tie2 antibody which prevents or treats diabetic macular edema, diabetic retinopathy, or critical limb ischaemia by binding to human Tie2 and activating human Tie2. [Solution] The inventors of the present invention studied anti-human Tie2 antibodies and have provided an anti-human Tie2 antibody including four heavy chain variable regions and four light chain variable regions. The heavy chain variable regions comprise an amino acid sequence from amino acid no. 1 to 122 of SEQ ID NO:2, and the light chain variable regions comprise an amino acid sequence from amino acid No. 1 to 113 of SEQ ID NO:4. One of the heavy chain variable regions and one of the light chain variable regions constitute one antigen-binding site, and the antibody includes four antigen-binding sites.

Description

technical field [0001] The present invention relates to novel anti-human Tie-2 antibodies. Background technique [0002] Tyrosine kinase with Ig and EGF homology domain 2 (Tie2) is a receptor-type tyrosine kinase. It is known that Tie2 is mainly expressed in vascular endothelial cells. As the ligands, known are angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), which are secreted glycoproteins of multimer type. [0003] Ang-1 acts as an agonist for Tie2. It has been found that when Tie2 binds to Ang-1, it is autophosphorylated by forming multimers and sends a signal into the cell, thereby promoting anti-apoptosis of vascular endothelial cells, vasculature via vascular permeability inhibition Stabilization, maturation and remodeling (Cell, 1996, Vol.87, pp.1171-1180; GenesDev., 1994, Vol.8, pp.1897-1909; Science, 1999, Vol.286, pp.2511- 2514; and Nat. Struct. Biol., 2003, Vol. 10, pp. 38-44). In addition, it is also known that Ang-1 exerts vasodilation and blood flow enh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P7/10A61P9/10A61P43/00C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12P21/08
CPCC12N2510/02C07K16/2863C07K2317/24C07K2317/33C07K2317/34C07K2317/52C07K2317/55C07K2317/64C07K2317/92C07K2319/00C07K2317/567C07K2317/75A61K2039/505C07K16/40C07K2317/35A61P27/02A61P43/00A61P7/10A61P9/10A61K39/395A61K39/3955C07K16/28C07K16/2896C07K2317/40C07K2317/51C07K2317/515C07K2317/565C07K2317/76C12N5/06C12N15/09C12N15/63
Inventor 蒲原正纯八木繁典石井芳则奈良裕美
Owner ASTELLAS PHARMA INC
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